Treatment of cancer remains a major problem in health care. One proposed strategy for treatment of cancer is inhibition of angiogenesis and thus inhibiting the generation and development of blood vessels, which supply the tumor with growth relevant means. A second strategy is direct inhibition of specific receptors on tumor cell surfaces, such as the inhibition of Her2 by Herceptin® or inhibition of EGFR by cetuximab (Erbitux®).
Inhibitors of integrins are considered to be potentially useful anti-tumor agents, because integrins are expressed on tumor neovasculature and mediate angiogenesis. In addition, integrins are expressed on certain tumor cells and may directly promote tumor growth and survival.
Integrins have no enzymatic activity, but integrins capable of binding their ligands (ligand-competent integrins) are activated by binding to proteins of the ECM. Integrins on one hand trigger intracellular kinase cascades to modulate cell growth and survival, and on the other associate with the cytoskeleton to drive cell attachment and locomotion. αvβ5 integrin specifically binds vitronectin, while αvβ3 also binds other macromolecules of the provisional ECM. αvβ3 was first noted in cancer as a progression-dependent marker on malignant melanoma. It enhanced melanoma growth in vivo and survival in vitro. αvβ3 blockers reversed these effects. Subsequently, αvβ3 was found in other tumors including glioblastoma, renal carcinomas, ovarian carcinomas and others. αvβ3 was also widely over-expressed in the ECs in many malignancies. In vitro, angiogenic models activated by tumor-derived growth factors over-expressed and required αvβ3 on the sprouting vasculature, while αvβ3 and αvβ5 blockade could suppress the angiogenic phenotype. αvβ5 was also shown to support the neo-vasculature induced by some tumor-derived growth factors. Inhibiting αvβ5 can trigger apoptosis of tumor cells.
The integrin receptors are over-expressed on tumor invasive blood vessels, on melanomas and some other malignancies, and modulate cellular response to growth factors. The vascular compartment is a promising therapeutic target because solid tumors depends on blood vessels for oxygen, nutrition, detoxification and the dispersion of blood borne metastases; the switch to the angiogenic phenotype marks a discrete step in the induction of malignancy, which is amenable to therapeutic intervention; and the vasculature undergoes continuous change, yet endothelial cells have genomic stability relative to the tumor, and are less likely to become drug-resistant through mutation.
A first anti-integrin drug is cilengitide and is considered to be a useful antitumor agent (Eskens F A, et al. (2003) Eur J Cancer 39:917-26). However, cilengitide is a small molecule that must be dosed frequently. The structure of cilengitide; selected salt form e.g. are disclosed in EP0770622, WO 0015244 and PCT/us07/01446 and are disclosed herein by reference.
A second anti-integrin drug is mouse mAb 17E6 (EMD 73034), that inhibits specifically the αv integrin subunit of human integrin receptor bearing cells. The mouse IgG1 antibody is described, for example by Mitjans et al. (1995; J. Cell Sci. 108, 2825) and U.S. Pat. No. 5,985,278 and EP 719 859. The complete variable heavy and light chain sequences are depicted in SEQ ID Nos. 27 and 28 (FIGS. 20 A, B). Murine 17E6 was generated from mice immunized with purified and Sepharose-immobilized human αvβ3. Spleen lymphocytes from immunized mice were fused with murine myeloma cells and one of the resulting hybridoma clones produced monoclonal antibody 17E6 (EMD 73034). Mouse mAb 17E6 is produced by hybridoma cell line 272-17E6 and deposited under accession number DSM ACC2160.
Mouse 17E6 antagonizes integrin interaction with the extracellular matrix (ECM), and perturbs the function of endothelial and tumor cells. Primary effects of the antibody include disrupting endothelial cell (EC) adhesion and movement, inducing their apoptosis, and suppressing the activation of growth factor pathways. Blockade by said antibody directly suppresses survival of both the activated endothelial cells and some tumor cells.
Monoclonal antibodies such as 17E6 are generally useful for the inhibition of extracellular protein-protein interactions, such as the inhibition of ligand-receptor interactions. However, monoclonal antibodies are often difficult to express and often provoke an immune response, such as an anti-idiotypic response, which limits their effectiveness.
These data and principal knowledge gathered so far supported the need of development of a modified mouse 17E6 antibody with improved properties that binds specifically to integrins, can be efficiently expressed, and is relatively non-immunogenic in humans as a therapeutic agent in cancer. Such an engineered antibody should have the potential to suppress the development of the tumor both indirectly, via the tumor vasculature, and directly on the tumor cells themselves.